[Show abstract][Hide abstract] ABSTRACT: The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the geno-mic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cyto-plasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cyto-plasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectrosco-py, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cy-toplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection.
[Show abstract][Hide abstract] ABSTRACT: The Pr55 Gag of HIV-1 orchestrates viral particle assembly in producer cells, which requires the genomic RNA and a lipid membrane as scaffolding platforms. The nucleocapsid (NC) domain with its two invariant CCHC zinc fingers flanked by unfolded basic sequences is thought to direct genomic RNA selection, dimerization and packaging during virus assembly. To further investigate the role of NC domain, we analyzed the assembly of Gag with deletions in the NC domain in parallel with that of wild-type Gag using fluorescence lifetime imaging microscopy (FLIM) combined with Förster resonance energy transfer (FRET) in HeLa cells. We found that upon binding to nucleic acids, the NC domain promotes the formation of compact Gag oligomers in the cytoplasm. Moreover, the intracellular distribution of the population of oligomers further suggests that oligomers progressively assemble during their trafficking towards the plasma membrane (PM), but with no dramatic changes in their compact arrangement. This ultimately results in the accumulation at the PM of closely packed Gag oligomers that likely arrange in hexameric lattices, as revealed by the perfect match between the experimental FRET value with the one calculated from the structural model of Gag in immature viruses. The distal finger and flanking basic sequences, but not the proximal finger appear to be essential for Gag oligomer compaction and membrane binding. Moreover, the full NC domain was found to be instrumental in the kinetics of Gag oligomerization and intracellular trafficking. These findings further highlight the key roles played by the NC domain in virus assembly.
Full-text · Article · Jan 2015 · Journal of Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The majority of the human immunodeficiency virus type 1 (HIV-1) proteins are able to self assemble into oligomers. Since these oligomers generally exhibit functions that differ from those of their monomeric counterpart, the regulation of the monomer-oligomer equilibria plays a central role in the viral cycle. To characterize the oligomerization of these proteins in live cells, the combination of fluorescence lifetime imaging microscopy (FLIM) with Förster resonance energy transfer (FRET) has proven to be very powerful. In this review, we illustrate the application of FRET-FLIM on the characterization of the oligomerization of the Vpr, Vif and Pr55Gag proteins of HIV-1 in fusion with eGFP and mCherry. For Vpr and Pr55Gag proteins, very high levels of FRET leading to strong decreases in eGFP fluorescence lifetime are obtained, as a consequence of the rather small size of the viral proteins, the strong packing of the protomers and the presence of multiple acceptors for one donor. Analyzing the time-resolved decays by a two-component analysis further provides the possibility to discriminate monomers from oligomers and to monitor the spatiotemporal evolution of both populations in the cells. Though FRET-FLIM unambiguously reveals the oligomerization of a given protein, it hardly discloses the oligomer stoichiometry (number of protomers per oligomers). This parameter can be obtained by fluorescence correlation spectroscopy, which allows further interpreting the FRET-FLIM data. FRET-FLIM is also highly useful to identify the determinants of the oligomerization process and to investigate its regulation by other HIV-1 proteins and host proteins.
No preview · Article · Jan 2015 · Springer Series in Chemical Physics
[Show abstract][Hide abstract] ABSTRACT: The HIV-1 Gag polyprotein precursor composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains orchestrates virus assembly via interactions between MA and the cell plasma membrane (PM) on one hand and NC and the genomic RNA on the other hand. As the Gag precursor can adopt a bent conformation, a potential interaction of the NC domain with the PM cannot be excluded during Gag assembly at the PM. To investigate the possible interaction of NC with lipid membranes in the absence of any interference from the other domains of Gag, we quantitatively characterized by fluorescence spectroscopy the binding of the mature NC protein to large unilamellar vesicles (LUVs) used as membrane models. We found that NC, either in its free form or bound to an oligonucleotide, was binding with high affinity (similar to 10(7) M-1) to negatively charged LUVs. The number of NC binding sites, but not the binding constant, was observed to decrease with the percentage of negatively charged lipids in the LUV composition, suggesting that NC and NC/oligonucleotide complexes were able to recruit negatively charged lipids to ensure optimal binding. However, in contrast to MA, NC did not exhibit a preference for phosphatidylinositol-(4,5)-bisphosphate. These results lead us to propose a modified Gag assembly model where the NC domain contributes to the initial binding of the bent form of Gag to the PM.
Full-text · Article · Nov 2014 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The Gag precursor of HIV-1, formed of the four proteic regions matrix (MA), capsid (CA), nucleocapsid (NC) and p6, orchestrates virus morphogenesis. This complex process relies on three major interactions, NC-RNA acting as a scaffold, CA-CA and MA-membrane that targets assembly to the plasma membrane (PM). The characterization of the molecular mechanism of retroviral assembly has extensively benefited from biochemical studies and more recently an important step forward was achieved with the use of fluorescence-based techniques and fluorescently-labelled viral proteins. In this review, we summarize the findings obtained with such techniques, notably quantitative-based approaches, which highlight the role of the NC region in Gag assembly.
[Show abstract][Hide abstract] ABSTRACT: This review aims at briefly presenting a retrospect on the retroviral nucleocapsid protein (NC), from an unspecific nucleic acid binding protein (NABP) to an all-in-one viral protein with multiple key functions in the early and late phases of the retrovirus replication cycle, notably reverse transcription of the genomic RNA and viral DNA integration into the host genome, and selection of the genomic RNA together with the initial steps of virus morphogenesis, respectively. In this context we will discuss the notion that NC protein has a flexible conformation and is thus a member of the growing family of intrinsically disordered proteins (IDP) where disorder may account, at least in part, for its functions as a nucleic acid (NA) chaperone and possibly as a protein chaperone vis à vis the viral DNA polymerase during reverse transcription. Lastly, we will briefly review the development of new anti-retroviral/AIDS compounds targeting HIV-1 NC because it represents an ideal target due to its multiple roles in the early and late phases of virus replication and its high degree of conservation.
[Show abstract][Hide abstract] ABSTRACT: The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein
and, more specifically, its (44–61) domain were recently shown to promote the annealing of complementary DNA sequences representing
the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic
mechanism of the basic Tat(44–61) peptide in this annealing further revealed that this peptide constitutes a representative
nucleic acid annealer. To further understand the structure–activity relationships of this highly conserved domain, we investigated
by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44–61) mutants. Our data
showed that the Tyr47 and basic residues of the Tat(44–61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions,
respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates
with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient
and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the
late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion.
Full-text · Article · Oct 2013 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in non-permissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (A3G) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55Gag. Recently, we showed that oligomerization of Vif into high-molecular mass complexes induces Vif folding and influences its binding to high affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays (FRET/FLIM) to investigate Vif-Vif interactions in living cells. By using two N-terminal tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain (161PPLP164) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate to Vif oligomerization. When co-expressed together with Pr55Gag, Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.
[Show abstract][Hide abstract] ABSTRACT: Two-photon excited microscopy has evolved as a routine technique for long-term cellular and in vivo imaging and is now available in most optical microscopy facilities. Classical dyes and fluorescent proteins, developed for epifluorescence or confocal microscopy are used, but unfortunately they present a low efficiency upon two-photon excitation inducing the need of high excitation power (over 20 mW). To reduce this excitation power, new dyes need to be developed allowing really low two-photon excitation power in the mW or sub mW range. We report here the conception, synthesis, physicochemical and photophysical properties of new functionalized diketopyrrolopyrrole (DPP) derivatives acting a fluorescent tags for biomolecules. They present high two-photon absorption cross-sections and bright luminescence around 600 nm. These two-photon optimized fluorophorophores were bio-conjugated to HIV-I Tat (44-61) and their cellular localization observed by two-photon excited microscopy using sub mW laser excitation power.
[Show abstract][Hide abstract] ABSTRACT: The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in non-permissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (A3G) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular mass complexes induces Vif folding and influences its binding to high affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays (FRET/FLIM) to investigate Vif-Vif interactions in living cells. By using two N-terminal tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate to Vif oligomerization. When co-expressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.
Full-text · Article · Apr 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is
assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through
pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences
corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the
oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into
the NCp7 molecular mechanism in reverse transcription.
No preview · Article · Feb 2013 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: HIV-1 particle assembly is driven by oligomerization of the Gag polyprotein precursor in infected cells. Translation of the full-length viral RNA results in the synthesis of large amounts of Gag molecules which oligomerize upon the combined interactions of its C-terminal NC domain with the genomic RNA and
of its N-terminal myristate and matrix basic residues with cellular membranes. HIV assembly has been studied during the past two decades mostly by means of biochemical techniques on transfected or infected cells as well as in vitro using purified Gag molecules. More recently, understanding the mechanisms of viral
assembly moved a big step forward due to the utilization of fluorescently labeled viral proteins, notably Gag, and of fluorescent microscopy techniques able to track single viral particles. In this chapter, we will summarize recent imaging data on HIV-1 Gag assembly at the level of the plasma membrane where viral particles bud in the form of cell-free viruses or can be transmitted to adjacent naı¨ve cells through
[Show abstract][Hide abstract] ABSTRACT: HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using (13)C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome.
[Show abstract][Hide abstract] ABSTRACT: The core protein of hepatitis c virus (HCV) is a structural protein with potent RNA chaperoning activities mediated by its
hydrophilic N-terminal domain D1, which is thought to play a key role in HCV replication. To further characterize the core
chaperoning properties, we studied the interactions between core D1 and the conserved HCV 3′X genomic region required for
genome replication. To this end, we monitored the real-time annealing kinetics of native and mutated fluorescently labelled
16-nt palindromic sequence (DLS) and 27-nt Stem Loop II (SL2) from X with their respective complementary sequences. Core D1
and peptides consisting of the core basic domains were found to promote both annealing reactions and partly switch the loop–loop
interaction pathway, which predominates in the absence of peptide, towards a pathway involving the stem termini. The chaperone
properties of the core D1 peptides were found to be mediated through interaction of their basic clusters with the oligonucleotide
phosphate groups, in line with the absence of high affinity site for core on HCV genomic RNA. The core ability to facilitate
the interconversion between different RNA structures may explain how this protein regulates RNA structural transitions during
Full-text · Article · Nov 2011 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52-96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52-96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52-96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4°C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52-96) peptide was stable in cells for at least 48h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.
[Show abstract][Hide abstract] ABSTRACT: Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand
transfer corresponds to the annealing of the (−) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned
by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(−)PBS annealing mechanism by activating a loop–loop kissing pathway
that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes,
we investigated the time-resolved fluorescence parameters of a (−)PBS derivative containing the fluorescent nucleoside analogue
2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(−)PBS annealing towards the loop pathway was associated
to a drastic restriction of the local DNA dynamics, indicating that NCp7 can ‘freeze’ PBS conformations competent for annealing
via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were
found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc
fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal
role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.
Full-text · Article · May 2011 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: During formation of HIV particles, the Gag polyproteins are thought to interact with Vpr proteins to promote their encapsidation in the nascent particles. To directly visualize and monitor the formation of the Gag-Vpr complexes and correlate their formation with Vpr oligomerization, we used two photon lifetime imaging microscopy (FLIM) and time laps microscopy on HeLa cells expressing eGFP-labelled Vpr and tetracystein-tagged Gag proteins detected by the biarsenical ReAsH labelling reagent. Using these fluorescent microscopy approaches, we found that Gag proteins interact directly with Vpr proteins, which results in their transfer from the nuclear to the plasma membrane. Vpr oligomerization was found critical for both its interaction with Gag proteins and its transfer to the plasma membrane. Moreover, mutations in the C-terminal domain of Gag abolished the interaction with Vpr and its redistribution to the plasma membrane, indicating that this domain was critical for interaction with Vpr. Taken together, these data show that the Gag polyproteins interact through their C-terminal domain with Vpr oligomers, promoting the redistribution of the latter at the plasma membrane and probably their incorporation into nascent viral particles.
No preview · Article · Feb 2011 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: The viral protein r (Vpr) of HIV-1 binds several host proteins leading to pleiotropic functions, such as G2/M cell cycle arrest, apoptosis induction and gene transactivation. Vpr is encapsidated through the Gag C-terminus into the nascent viral particles, suggesting that Vpr plays several important functions in the early stages of the viral lifecycle. In this regard, Vpr interacts with nucleic acids and membranes to facilitate the preintegration complex migration and incorporation into the nucleus of nondividing cells. Thus, Vpr has to recruit several host and viral factors to promote its functions during HIV-1 pathogenesis. This article focuses on its interacting partners by giving an overview of the functional outcome of the different Vpr complexes, as well as the structural determinants of Vpr required for its binding properties.
[Show abstract][Hide abstract] ABSTRACT: The nucleocapsid protein (NCp7) is a major HIV-1 structural protein that plays key roles in viral replication, mainly through its conserved zinc fingers that direct specific interactions with the viral nucleic acids. Owing to its high degree of conservation and critical functions, NCp7 represents a target of choice for drugs that can potentially complement HAART, thus possibly impairing the circulation of drug-resistant HIV-1 strains. Zinc ejectors showing potent antiretroviral activity were developed, but early generations suffered from limited selectively and significant toxicity. Compounds with improved selectivity have been developed and are being explored as topical microbicide candidates. Several classes of molecules inhibiting the interaction of NCp7 with the viral nucleic acids have also been developed. Although small molecules would be more suited for drug development, most molecules selected by screening showed limited antiretroviral activity. Peptides and RNA aptamers appear to be more promising, but the mechanism of their antiretroviral activity remains elusive. Substantial and more concerted efforts are needed to further develop anti-HIV drugs targeting NCp7 and bring them to the clinic.
[Show abstract][Hide abstract] ABSTRACT: The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting
with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties.
To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic
clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1
transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing
kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but
was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated
through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate
that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs,
which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone
properties are mostly supported by its basic clusters.
Full-text · Article · Feb 2010 · Nucleic Acids Research